Functional selectivity of insulin receptor revealed by aptamer-trapped receptor structures

Activation of insulin receptor (IR) initiates a cascade of conformational changes and autophosphorylation events. Herein, we determined three structures of IR trapped by aptamers using cryo-electron microscopy. The A62 agonist aptamer selectively activates metabolic signaling. In the absence of insulin, the two A62 aptamer agonists of IR adopt an insulin-accessible arrowhead conformation by mimicking site-1/site-2’ insulin coordination. Insulin binding at one site triggers conformational changes in one protomer, but this movement is blocked in the other protomer by A62 at the opposite site. A62 binding captures two unique conformations of IR with a similar stalk arrangement, which underlie Tyr1150 mono-phosphorylation (m-pY1150) and selective activation for metabolic signaling. The A43 aptamer, a positive allosteric modulator, binds at the opposite side of the insulin-binding module, and stabilizes the single insulin-bound IR structure that brings two FnIII-3 regions into closer proximity for full activation. Our results suggest that spatial proximity of the two FnIII-3 ends is important for m-pY1150, but multi-phosphorylation of IR requires additional conformational rearrangement of intracellular domains mediated by coordination between extracellular and transmembrane domains.


IR-A43(500nM)
--       experiments. c, d, Quantification of western blot data for m-pY1150 or pY1150/pY1151 shown in Supplementary Fig. 10b. Experiments were repeated three times independently and graphs show means ± standard deviation (n=3). e, Insulin-induced IR phosphorylation in CHO-K1 cells expressing WT IR or the IRlinker mutant. The data were representative of three independent experiments. f, g, Quantification of western blot data for m-pY1150 or pY1150/pY1151 shown in Supplementary Fig. 10e. Experiments were repeated three times independently and graphs show means ± standard deviation (n=3). Source data are provided as a Source Data file.

Description of the structures of aptamers
Throughout this work, the entire naphthyl-modified deoxyuridine nucleotide (5-[N-(1naphthylmethyl)carboxamide]-2'-deoxyuridine) is referred to as PX, the 2-napthyl moiety is 2NapX, and the uridine base is dUX (Supplementary Fig. 3a). The entire benzyl-modified deoxyuridine nucleotide (5-[N-benzylcarboxamide]-2'-deoxyuridine) is referred to as BX and the 5-benzyl moiety is 5BzX. The 2'-fluoro-modified bases are referred to as fX and 2'-O-methylmodified bases are referred to as mX. X is the nucleotide number.

Description of the A62 structure
The A62 aptamer consists of 25 nucleotides in which seven dTs (deoxythymidine) are substituted by three Ps and four Bs ( Supplementary Fig. 3a) 7 . A62 forms a non-helical compact structure, which is primarily stabilized through numerous base stacking interactions and Watson-Crick (WC) base pairs along with H-bonds and hydrophobic interactions (Fig. 2c).
At the lower part of A62, three successive WC base pairs (dA5-dB24, fC6-dG23, and dG7-fC22) are highlighted between the stem and the side loops (Fig. 2e, Supplementary Fig. 3c). Below the dA5-dB24 base pair, the dC1 to dB4 chain is positioned at the bottom with bases vertically oriented with respect to the dA5-dB24 bases. The 2Nap20 ring is sandwiched between the dA5-dB24 base pair and the dC1-dB4 chain, and the dU20 base is vertically oriented with respect to 2Nap20, forming a three-layer stack with B4(dU) and B24(Bz) at the back of 2Nap20 ( Supplementary Fig. 3c).
In the middle region, the 2Nap10 ring is stacked on top of the dG7-fC22 base pairs on one side, and also reciprocally stacked on the dA9 base on another side (Fig. 2e). The mG11 base is sandwiched between the dU10 and fA19 bases, forming a four-layer stack, which is vertically oriented with respect to the three WC base pairs at the lower part, which forces a significant bend in the aptamer. On top, the dU14 base occupies the center of the head loop, forming a three-layer stack with the mG13 and dP16 (2Nap) bases (Fig. 2e, Supplementary Fig. 4f). The 5Bz14 resides on top of the four-layer stack (dA9-dU10-mG11-fA19) and is perpendicularly packed against another four-layer stack formed by dU16, dA17, dG18, and fA12 at the side. The three base-base stacking interactions (dU14, mG13, and 2Nap16) on top force the C15 base to flip, allowing it to interact with IR (R14 and F64 at L1). Overall, several modified bases engage in hydrophobic, Hbond, and stacking interactions to stabilize A62 and allow the aptamer to interact with IR.

Interaction between A62 and IR
Due to their internal stacking, several bases are flipped and exposed in both side loops and a head loop, enabling A62 to interact with L1 and FnIII-1' of IR ( Fig. 2d-g). In the L1 site, loop H and L make contacts with L1-β2 (Fig. 2d, f). This interface is stabilized through extensive hydrophobic interactions, ion pairs, and H-bonds between A62 bases and IR residues. On the opposite side, a flat face is formed by the stem and two loops packed against the side of the main β-sheet of FnIII-1' through stacking between modified bases and β-sheet residues, and ion pairs between the A62 phosphate backbone and basic residues (Fig. 2d, g). In the FnIII-1'-loop S interface, three nucleotides (fA19, dP20, and mC21) bind to residues of FnIII-1', the dA19 base engages in hydrophobic interactions with L552, and phosphate groups of dP20 and mC21 interact with R488 and Q546, respectively. The L1-loop H interface involves docking of 2Nap16 into the surface pocket formed by F64, R65, F88, F89, F96, and R118 (Fig. 2f). Two residue-phosphate interactions (between dP16 and Tyr60 and Gln34, and between dA17 and R14) augment the interface (Fig. 2g). In the L1-loop L interface, the dA17 phosphate group binds to Y67, and the ribose ring of dA9 forms an H-bond with R65. In the FnIII-1'-stem interface, the dU3 base interacts with K544 and His548, and the dU4 base is reciprocally sandwiched between Y477 and dU20.

Interactions between A43 and IR
The major groove packs against the main β-sheet of FnIII-1', whereas the shallow groove on the opposite side and the stem region are exposed to the surface without contacting IR (Fig 3d,